cDNA cloning and expression of murine macrophage colony-stimulating factor from L929 cells.

A 4-kilobase and a 2-kilobase cDNA clone encoding a murine macrophage colony-stimulating factor have been isolated. Except for 2 amino acid residue differences, these two clones encode the same 520 amino acid residue protein, which is preceded by a 32-amino acid residue signal peptide. The two clones, whose molecular masses correspond to the two transcripts observed in murine L929 fibroblasts, contain 3' untranslated regions that are markedly different in sequence and length. Both clones can be expressed in COS cells and the recombinant protein is active in a mouse bone marrow colony assay.

Expression, Glycosylation, and Secretion of an Aspergillus Glucoamylase by Saccharomyces cerevisiae.

A strain of Saccharomyces cerevisiae capable of simultaneous hydrolysis and fermentation of highly polymerized starch oligosaccharides was constructed. The Aspergillus awamori glucoamylase enzyme, form GAI, was expressed in Saccharomyces cerevisiae by means of the promoter and termination regions from a yeast enolase gene. Yeast transformed with plasmids containing an intron-free recombinant glucoamylase gene efficiently secreted glucoamylase into the medium, permitting growth of the transformants on starch as the sole carbon source. The natural leader sequence of the precursor of glucoamylase (preglucoamylase) was processed correctly by yeast, and the secreted enzyme was glycosylated through both N- and O-linkages at levels comparable to the native Aspergillus enzyme. The data provide evidence for the utility of yeast as an organism for the production, glycosylation, and secretion of heterologous proteins.

Molecular cloning and characterization of the glucoamylase gene of Aspergillus awamori.

The filamentous ascomycete Aspergillus awamori secretes large amounts of glucoamylase upon growth in medium containing starch, glucose, or a variety of hexose sugars and sugar polymers. We examined the mechanism of this carbon source-dependent regulation of glucoamylase accumulation and found a several hundredfold increase in glucoamylase mRNA in cells grown on an inducing substrate, starch, relative to cells grown on a noninducing substrate, xylose. We postulate that induction of glucoamylase synthesis is regulated transcriptionally. Comparing total mRNA from cells grown on starch and xylose, we were able to identify an inducible 2.3-kilobase mRNA-encoding glucoamylase. The glucoamylase mRNA was purified and used to identify a molecularly cloned 3.4-kilobase EcoRI fragment containing the A. awamori glucoamylase gene. Comparison of the nucleotide sequence of the 3.4-kilobase EcoRI fragment with that of the glucoamylase I mRNA (as determined from molecularly cloned cDNA) revealed the existence of four intervening sequences within the glucoamylase gene. The 5' end of the glucoamylase mRNA was mapped to several locations within a region -52 to -73 nucleotides from the translational start. Sequence and structural features of the glucoamylase gene of the filamentous ascomycete A. awamori were examined and compared with those reported in genes of other eucaryotes.